The search for new therapeutic agents has been greatly aided in recent years by better understanding of the structure of enzymes and other biomolecules associated with target diseases. One important class of enzymes that has been the subject of extensive study is the protein kinases.
Protein kinases mediate intracellular signal transduction. They do this by effecting a phosphoryl transfer from a nucleoside triphosphate to a protein acceptor that is involved in a signaling pathway. There are a number of kinases and pathways through which extracellular and other stimuli cause a variety of cellular responses to occur inside the cell. Examples of such stimuli include environmental and chemical stress signals (e.g. osmotic shock, heat shock, ultraviolet radiation, bacterial endotoxin, H2O2), cytokines (e.g. interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α)), and growth factors (e.g. granulocyte macrophage-colony-stimulating factor (GM-CSF), and fibroblast growth factor (FGF). An extracellular stimulus may effect one or more cellular responses related to cell growth, migration, differentiation, secretion of hormones, activation of transcription factors, muscle contraction, glucose metabolism, control of protein synthesis and regulation of cell cycle.
Many diseases are associated with abnormal cellular responses triggered by protein kinase-mediated events. These diseases include autoimmune diseases, inflammatory diseases, neurological and neurodegenerative diseases, cancer, cardiovascular diseases, allergies and asthma, Alzheimer's disease or hormone-related diseases. Accordingly, there has been a substantial effort in medicinal chemistry to find protein kinase inhibitors that are effective as therapeutic agents. A challenge has been to find protein kinase inhibitors that act in a selective manner. Since there are numerable protein kinases that are involved in a variety of cellular responses, non-selective inhibitors may lead to unwanted side effects.
AKT (also known as PKB or Rac-PK beta), a serine/threonine protein kinase, has been shown to be overexpressed in several types of cancer and is a mediator of normal cell functions [(Khwaja, A., Nature, 401, pp. 33-34, 1999); (Yuan, Z. Q., et al., Oncogene, 19, pp. 2324-2330, 2000); (Namikawa, K., et al., J Neurosci., 20, pp. 2875-2886, 2000)]. AKT comprises an N-terminal pleckstrin homology (PH) domain, a kinase domain and a C-terminal “tail” region. Three isoforms of human AKT kinase (AKT-1, -2 and -3) have been reported so far [(Cheng, J. Q., Proc. Natl. Acad. Sci. USA, 89, pp. 9267-9271, 1992); (Brodbeck, D. et al., J. Biol. Chem. 274, pp. 9133-9136, 1999)]. The PH domain binds 3-phosphoinositides, which are synthesized by phosphatidyl inositol 3-kinase (PI3K) upon stimulation by growth factors such as platelet derived growth factor (PDGF), nerve growth factor (NGF) and insulin-like growth factor (IGF-1) [(Kulik et al., Mol. Cell. Biol., 17, pp. 1595-1606, 1997); (Hemmings, B. A., Science, 275, pp. 628-630, 1997)]. Lipid binding to the PH domain promotes translocation of AKT to the plasma membrane and facilitates phosphorylation by another PH-domain-containing protein kinases, PDK1 at Thr308, Thr309, and Thr305 for the AKT isoforms 1, 2 and 3, respectively. A second, as of yet unknown, kinase is required for the phosphorylation of Ser473, Ser474 or Ser472 in the C-terminal tails of AKT-1, -2 and -3 respectively, in order to yield a fully activated AKT enzyme.
Once localized to the membrane, AKT mediates several functions within the cell including the metabolic effects of insulin (Calera, M. R. et al., J. Biol. Chem., 273, pp. 7201-7204, 1998), induction of differentiation and/or proliferation, protein synthesisans stress responses (Alessi, D. R. et al., Curr. Opin. Genet. Dev., 8, pp. 55-62, 1998).
Manifestations of altered AKT regulation appear in both injury and disease, the most important role being in cancer. The first account of AKT was in association with human ovarian carcinomas where expression of AKT was found to be amplified in 15% of cases (Cheng, J. Q. et al., Proc. Natl. Acad. Sci. U.S.A., 89, pp. 9267-9271, 1992). It has also been found to be overexpressed in 12% of pancreatic cancers (Cheng, J. Q. et al., Proc. Natl. Acad. Sci. U.S.A., 93, pp. 3636-3641, 1996). It was demonstrated that AKT-2 was over-expressed in 12% of ovarian carcinomas and that amplification of AKT was especially frequent in 50% of undifferentiated tumours, showing that AKT is also associated with tumour aggressiveness (Bellacosa, et al., Int. J. Cancer, 64, pp. 280-285, 1995).
The 3-phosphoinositide-dependent protein kinase-1 (PDK1) plays a key role in regulating the activity of a number of kinases belonging to the AGC subfamily of protein kinases (Alessi, D. et al., Biochem. Soc. Trans, 29, pp. 1, 2001). These include isoforms of protein kinase B (PKB, also known as AKT), p70 ribosomal S6 kinase (S6K) (Avruch, J. et al., prog. Mol. Subcell. Biol., 2001, 26, pp. 115, 2001), and p90 ribosomal S6 kinase (Frödin, M. et al., EMBO J., 19, pp. 2924-2934, 2000). PDK1 mediated signaling is activated in response to insulin and growth factors and as a consequence of attachment of the cell to the extracellular matrix (integrin signaling). Once activated these enzymes mediate many diverse cellular events by phosphorylating key regulatory proteins that play important roles controlling processes such as cell survival, growth, proliferation and glucose regulation [(Lawlor, M. A. et al., J. Cell Sci., 114, pp. 2903-2910, 2001), (Lawlor, M. A. et al., EMBO J., 21, pp. 3728-3738, 2002)]. PDK1 is a 556 amino acid protein, with an N-terminal catalytic domain and a C-terminal pleckstrin homology (PH) domain, which activates its substrates by phosphorylating these kinases at their activation loop (Belham, C. et al., Curr. Biol., 9, pp. R93-R96, 1999). Many human cancers including prostate and NSCL have elevated PDK1 signaling pathway function resulting from a number of distinct genetic events such as PTEN mutations or over-expression of certain key regulatory proteins [(Graff, J. R., Expert Opin. Ther. Targets, 6, pp. 103-113, 2002), (Brognard, J., et al., Cancer Res., 61, pp. 3986-3997, 2001)] Inhibition of PDK1 as a mechanism to treat cancer was demonstrated by transfection of a PTEN negative human cancer cell line (U87MG) with antisense oligonucleotides directed against PDK1. The resulting decrease in PDK1 protein levels led to a reduction in cellular proliferation and survival (Flynn, P., et al., Curr. Biol., 10, pp. 1439-1442, 2000). Consequently the design of ATP binding site inhibitors of PDK1 offers, amongst other treatments, an attractive target for cancer chemotherapy.
The diverse range of cancer cell genotypes has been attributed to the manifestation of the following six essential alterations in cell physiology: self-sufficiency in growth signaling, evasion of apoptosis, insensitivity to growth-inhibitory signaling, limitless replicative potential, sustained angiogenesis, and tissue invasion leading to metastasis (Hanahan, D. et al., Cell, 100, pp. 57-70, 2000). PDK1 is a critical mediator of the PI3K signalling pathway, which regulates a multitude of cellular function including growth, proliferation and survival. Consequently inhibition of this pathway could affect four or more of the six defining requirements for cancer progression, as such it is anticipated that a PDK1 inhibitor will have an effect on the growth of a very wide range of human cancers.
Specifically, increased levels of PI3K pathway activity has been directly associated with the development of a number of human caners, progression to an aggressive refractory state (acquired resistance to chemotherapies) and poor prognosis. This increased activity has been attributed to a series of key events including decreased activity of negative pathway regulators such as the phosphatase PTEN, activating mutations of positive pathway regulators such as Ras, and overexpression of components of the pathway itself such as PKB, examples include: brain (gliomas), breast, colon, head and neck, kidney, lung, liver, melanoma, ovarian, pancreatic, prostate, sarcoma, thyroid [(Teng, D. H. et al., Cancer Res., 57, pp. 5221-5225, 1997), (Brognard, J. et al., Cancer Res., 61, pp. 3986-3997, 2001), (Cheng, J. Q. et al., Proc. Natl. Acad. Sci., 93, pp. 3636-3641, 1996), Int. J. Cancer, 64, pp. 280, 1995), (Graff, J. R., Expert Opin. Ther. Targets, 6, pp. 103-113, 2002), Am. J. Pathol., 159, pp. 431, 2001)].
Additionally, decreased pathway function through gene knockout, gene knockdown, dominant negative studies and small molecule inhibitors of the pathway have been demonstrated to reverse many of the cancer phenotypes in vitro (some studies have also demonstrated a similar effect in vivo) such as block proliferation, reduce viability and sensitize cancer cells to known chemotherapies in a series of cell lines, representing the following cancers: pancreatic [(Cheng, J. Q. et al., Proc. Natl. Acad. Sci., 93, pp. 3636-3641, 1996), Neoplasia, 3, pp. 278, 2001)], lung [(Brognard, J. et al., Cancer Res., 61, pp. 3986-3997, 2001), Neoplasia, 3, pp. 278, 2001)], ovarian [(Hayakawa, J. et al., Cancer Res., 60, pp. 5988-5994, 2000), Neoplasia, 3, pp. 278, 2001)], breast (Mol. Cancer Ther., 1, pp. 707, 2002), colon [(Neoplasia, 3, pp. 278, 2001), (Arico, S. et al., J. Biol. Chem., 277, pp. 27613-27621, 2002)], cervical (Neoplasia, 3, pp. 278, 2001), prostate [(Endocrinology, 142, pp. 4795, 2001), (Thakkar, H. et al. J. Biol. Chem., 276, pp. 38361-38369, 2001), (Chen, X. et al., Oncogene, 20, pp. 6073-6083, 2001)] and brain (glioblastomas) [(Flynn, P. et al., Curr. Biol., 10, pp. 1439-1442, 2000)].
Accordingly, there is a great need to develop inhibitors of AKT and PDK1 protein kinases that are useful in treating various diseases or conditions associated with AKT and PDK1 activation, particularly given the inadequate treatments currently available for the majority of these disorders.